Quantitative in situ liquid cell transmission electron microscopy of microtubules

Microtubules form the basic structure, and transport systems in cells, responsible for fundamental processes including cell division, and hence are important drug targets for diseases including cancer, with common taxane-based drugs having them as their biological target of action. Broadly, one of the mysteries of cell biology is the basic set of properties involved in the seed and growth of microtubules and the processes underlying how these systems are initiated. New avenues for controlling cell development and division, possibly related to anticancer treatments will come with a fundamental understanding of the basic mechanisms that lead to unusual switching behavior – dynamic instability. After decades of studies, both in cells and in reconstituted systems there are many open questions about this fascinating behavior. The major bottleneck in exploring microtubules is that the process of seeding is short-lived and the molecular species are structurally very small making standard microscopy observations very difficult, especially for capturing dynamics. In situ liquid cell transmission electron microscopy (LCTEM) has the potential to solve this crucial problem. To enable these kinds of experiments, the microtubule growth, and concentration need to be optimized for confinement within the liquid-cell. Imaging conditions need to be optimized to maximize contrast (as biological systems produce very low contrast under electron beam). We contend that LCTEM experiments replicating naturally occurring nucleation, growth, shrinkage, and re-growth process of microtubules and quantitative image analysis would offer us unique insights into their formation and reorganization.